Improved flow cytometric analysis of the budding yeast cell cycle

The budding yeast, Saccharomyces cerevisiae has been a remarkably useful model system for the study of eukaryotic cell cycle regulation. Flow cytometric analysis of DNA content in budding yeast has become a standard tool for the analysis of cell cycle progression. However, popular protocols utilizing the DNA binding dye, propidium iodide, suffer from a number of drawbacks that confound accurate analysis by flow cytometry. Here we show the utility of the DNA binding dye, SYTOX Green, in the cell cycle analysis of yeast. Samples analyzed using SYTOX Green exhibited better coefficients of variation, improved linearity between DNA content and fluorescence, and decreased peak drift associated with changes in dye concentration, growth conditions or cell size.     

Metabolic control of cytosolic‐facing pools of diacylglycerol in budding yeast

Diacylglycerol (DAG) is a key signaling lipid and intermediate in lipid metabolism. Our knowledge of DAG distribution and dynamics in cell membranes is limited. Using live‐cell fluorescence microscopy we investigated the localization of yeast cytosolic‐facing pools of DAG in response to conditions where lipid homeostasis and DAG levels were known to be altered. Two main pools were monitored over time using DAG sensors. One pool was associated with vacuolar membranes and the other localized to sites of polarized growth. Dynamic changes in DAG distribution were observed during resumption of growth from stationary phase, when DAG is used to support phospholipid synthesis for membrane proliferation. Vacuolar membranes experienced constant morphological changes displaying DAG enriched microdomains coexisting with liquid‐disordered areas demarcated by Vph1. Formation of these domains was dependent on triacylglycerol (TAG) lipolysis. DAG domains and puncta were closely connected to lipid droplets. Lack of conversion of DAG to phosphatidate in growth conditions dependent on TAG mobilization, led to the accumulation of DAG in a vacuolar‐associated compartment, impacting the polarized distribution of DAG at budding sites. DAG polarization was also regulated by phosphatidylserine synthesis/traffic and sphingolipid synthesis in the Golgi.      

Understanding the physics of oligonucleotide microarrays: the Affymetrix spike-in data reanalysed

The Affymetrix U95 and U133 Latin-Square spike-in datasets are reanalysed, together with a dataset from a version of the U95 spike-in experiment without a complex non-specific background. The approach uses a physico-chemical model which includes the effects of the specific and non-specific hybridization and probe folding at the microarray surface, target folding and hybridization in the bulk RNA target solution and duplex dissociation during the post-hybridization washing phase. The model predicts a three-parameter hyperbolic response function that fits well with fluorescence intensity data from all the three datasets. The importance of the various hybridization and washing effects in determining each of the three parameters is examined, and some guidance is given as to how a practical algorithm for determining specific target concentrations might be developed.     

Molecular analysis of kinetochore-microtubule attachment in budding yeast

The complex series of movements that mediates chromosome segregation during mitosis is dependent on the attachment of microtubules to kinetochores, DNA-protein complexes that assemble on centromeric DNA. We describe the use of live-cell imaging and chromatin immunoprecipitation in S. cerevisiae to identify ten kinetochore subunits, among which are yeast homologs of microtubule binding proteins in animal cells. By analyzing conditional mutations in several of these proteins, we show that they are required for the imposition of tension on paired sister kinetochores and for correct chromosome movement. The proteins include both molecular motors and microtubule associated proteins (MAPs), implying that motors and MAPs function together in binding chromosomes to spindle microtubules.     

Logical analysis of the budding yeast cell cycle

The budding yeast Saccharomyces cerevisiae is a model organism that is commonly used to investigate control of the eukaryotic cell cycle. Moreover, because of the extensive experimental data on wild type and mutant phenotypes, it is also particularly suitable for mathematical modelling and analysis. Here, I present a new Boolean model of the budding yeast cell cycle. This model is consistent with a wide range of wild type and mutant phenotypes and shows remarkable robustness against perturbations, both to reaction times and the states of component genes/proteins. Because of its simple logical nature, the model is suitable for sub-network analysis, which can be used to identify a four node core regulatory circuit underlying cell cycle regulation. Sub-network analysis can also be used to identify key sub-dynamics that are essential for viable cell cycle control, as well as identifying the sub-dynamics that are most variable between different mutants.     

Apn1 and Apn2 endonucleases prevent accumulation of repair-associated DNA breaks in budding yeast as revealed by direct chromosomal analysis

Base excision repair (BER) provides relief from many DNA lesions. While BER enzymes have been characterized biochemically, BER functions within cells are much less understood, in part because replication bypass and double-strand break (DSB) repair can also impact resistance to base damage. To investigate BER in vivo, we examined the repair of methyl methanesulfonate (MMS) induced DNA damage in haploid G1 yeast cells, so that replication bypass and recombinational DSB repair cannot occur. Based on the heat-lability of MMS-induced base damage, an assay was developed that monitors secondary breaks in full-length yeast chromosomes where closely spaced breaks yield DSBs that are observed by pulsed-field gel electrophoresis. The assay detects damaged bases and abasic (AP) sites as heat-dependent breaks as well as intermediate heat-independent breaks that arise during BER. Using a circular chromosome, lesion frequency and repair kinetics could be easily determined. Monitoring BER in single and multiple glycosylase and AP-endonuclease mutants confirmed that Mag1 is the major enzyme that removes MMS-damaged bases. This approach provided direct physical evidence that Apn1 and Apn2 not only repair cellular base damage but also prevent break accumulation that can result from AP sites being channeled into other BER pathway(s).     

Contact spotting of protein microarrays coupled with spike-in of normalizer protein permits time-resolved analysis of ERBB receptor signaling

Protein microarrays allow highly accurate comparison and quantification of numerous biological samples in parallel while requiring only little material. This qualifies protein arrays for systems biology and clinical research where only limited sample material is available, but a precise readout is required. With the introduction of signal normalization steps to monitor the drop size of manually contact-spotted RP protein arrays, the usefulness of normalizer proteins to ensure a high-throughput but inexpensive protein analysis was demonstrated. This approach was applied for the analysis of signaling through ERBB receptor activated kinases in the breast cancer cell line MCF-7. Activation of ERK1/2 and AKT by ERBB1 (EGFR), ERRB2 (HER2/neu), and ERBB3-4 was monitored in a time-resolved manner. Analysis of pathway activation by stimulation with epidermal growth factor and heregulin, or inhibition by blocking with gefitinib or herceptin allowed a characterization of the distinct signaling properties of the different ERBB receptor subtypes.     

Automated image analysis of protein localization in budding yeast

MOTIVATION: The yeast Saccharomyces cerevisiae is the first eukaryotic organism to have its genome completely sequenced. Since then, several large-scale analyses of the yeast genome have provided extensive functional annotations of individual genes and proteins. One fundamental property of a protein is its subcellular localization, which provides critical information about how this protein works in a cell. An important project therefore was the creation of the yeast GFP fusion localization database by the University of California, San Francisco, USA (UCSF). This database provides localization data for 75% of the proteins believed to be encoded by the yeast genome. These proteins were classified into 22 distinct subcellular location categories by visual examination. Based on our past success at building automated systems to classify subcellular location patterns in mammalian cells, we sought to create a similar system for yeast. RESULTS: We developed computational methods to automatically analyze the images created by the UCSF yeast GFP fusion localization project. The system was trained to recognize the same location categories that were used in that study. We applied the system to 2640 images, and the system gave the same label as the previous assignments to 2139 images (81%). When only the highest confidence assignments were considered, 94.7% agreement was observed. Visual examination of the proteins for which the two approaches disagree suggests that at least some of the automated assignments may be more accurate. The automated method provides an objective, quantitative and repeatable assignment of protein locations that can be applied to new collections of yeast images (e.g. for different strains or the same strain under different conditions). It is also important to note that this performance could be achieved without requiring colocalization with any marker proteins. AVAILABILITY: The original images analyzed in this article are available at http://yeastgfp.ucsf.edu, and source code and results are available at http://murphylab.web.cmu.edu/software.     

Integrative analysis of cell cycle control in budding yeast

The adaptive responses of a living cell to internal and external signals are controlled by networks of proteins whose interactions are so complex that the functional integration of the network cannot be comprehended by intuitive reasoning alone. Mathematical modeling, based on biochemical rate equations, provides a rigorous and reliable tool for unraveling the complexities of molecular regulatory networks. The budding yeast cell cycle is a challenging test case for this approach, because the control system is known in exquisite detail and its function is constrained by the phenotypic properties of >100 genetically engineered strains. We show that a mathematical model built on a consensus picture of this control system is largely successful in explaining the phenotypes of mutants described so far. A few inconsistencies between the model and experiments indicate aspects of the mechanism that require revision. In addition, the model allows one to frame and critique hypotheses about how the division cycle is regulated in wild-type and mutant cells, to predict the phenotypes of new mutant combinations, and to estimate the effective values of biochemical rate constants that are difficult to measure directly in vivo.     

Comparative analysis of cytokinesis in budding yeast, fission yeast and animal cells

Cytokinesis is a temporally and spatially regulated process through which the cellular constituents of the mother cell are partitioned into two daughter cells, permitting an increase in cell number. When cytokinesis occurs in a polarized cell it can create daughters with distinct fates. In eukaryotes, cytokinesis is carried out by the coordinated action of a cortical actomyosin contractile ring and targeted membrane deposition. Recent use of model organisms with facile genetics and improved light-microscopy methods has led to the identification and functional characterization of many proteins involved in cytokinesis. To date, this analysis indicates that some of the basic components involved in cytokinesis are conserved from yeast to humans, although their organization into functional machinery that drives cytokinesis and the associated regulatory mechanisms bear species-specific features. Here, we briefly review the current status of knowledge of cytokinesis in the budding yeast Saccharomyces cerevisiae, the fission yeast Schizosaccharomyces pombe and animal cells, in an attempt to highlight both the common and the unique features. Although these organisms diverged from a common ancestor about a billion years ago, there are eukaryotes that are far more divergent. To evaluate the overall evolutionary conservation of cytokinesis, it will be necessary to include representatives of these divergent branches. Nevertheless, the three species discussed here provide substantial mechanistic diversity.     

A genomic analysis of chronological longevity factors in budding yeast

Chronological life span (CLS) has been studied as an aging paradigm in yeast. A few conserved aging genes have been identified that modulate both chronological and replicative longevity in yeast as well as longevity in the nematode Caenorhabditis elegans; however, a comprehensive analysis of the relationship between genetic control of chronological longevity and aging in other model systems has yet to be reported. To address this question, we performed a functional genomic analysis of chronological longevity for 550 single-gene deletion strains, which accounts for approximately 12% of the viable homozygous diploid deletion strains in the yeast ORF deletion collection. This study identified 33 previously unknown determinants of CLS. We found no significant enrichment for enhanced CLS among deletions corresponding to yeast orthologs of worm aging genes or among replicatively long-lived deletion strains, although a trend toward overlap was noted. In contrast, a subset of gene deletions identified from a screen for reduced acidification of culture media during growth to stationary phase was enriched for increased CLS. These results suggest that genetic control of CLS under the most commonly utilized assay conditions does not strongly overlap with longevity determinants in C. elegans, with the existing confined to a small number of genetic pathways. These data also further support the model that acidification of the culture medium plays an important role in survival during chronological aging in synthetic medium, and suggest that chronological aging studies using alternate medium conditions may be more informative with regard to aging of multicellular eukaryotes.Key words: aging, genomic, screen, lifespan, yeast, C. elegans, pH, chronological, replicative     

Common gene expression strategies revealed by genome-wide analysis in yeast

Background  

Gene expression is a two-step synthesis process that ends with the necessary amount of each protein required to perform its function. Since the protein is the final product, the main focus of gene regulation should be centered on it. However, because mRNA is an intermediate step and the amounts of both mRNA and protein are controlled by their synthesis and degradation rates, the desired amount of protein can be achieved following different strategies.     

A genomic analysis of chronological longevity factors in budding yeast

Chronological life span (CLS) has been studied as an aging paradigm in yeast. A few conserved aging genes have been identified that modulate both chronological and replicative longevity in yeast as well as longevity in the nematode Caenorhabditis elegans; however, a comprehensive analysis of the relationship between genetic control of chronological longevity and aging in other model systems has yet to be reported. To address this question, we performed a functional genomic analysis of chronological longevity for 550 single-gene deletion strains, which accounts for approximately 12% of the viable homozygous diploid deletion strains in the yeast ORF deletion collection. This study identified 33 previously unknown determinants of CLS. We found no significant enrichment for enhanced CLS among deletions corresponding to yeast orthologs of worm aging genes or among replicatively long-lived deletion strains, although a trend toward overlap was noted. In contrast, a subset of gene deletions identified from a screen for reduced acidification of culture media during growth to stationary phase was enriched for increased CLS. These results suggest that genetic control of CLS under the most commonly utilized assay conditions does not strongly overlap with longevity determinants in C. elegans, with the existing confined to a small number of genetic pathways. These data also further support the model that acidification of the culture medium plays an important role in survival during chronological aging in synthetic medium, and suggest that chronological aging studies using alternate medium conditions may be more informative with regard to aging of multicellular eukaryotes.     

Structural and SAXS analysis of the budding yeast SHU-complex proteins

In Saccharomyces cerevisiae, four proteins, Shu1, Shu2, Psy3 and Csm2, form a stable SHU-complex both in vivo and in vitro. These proteins are involved in the early stages of the homologous recombination DNA damage repair process. In this paper, the crystal structure of the Psy3–Csm2 sub-complex is presented at 1.8 Å resolution and successfully fitted into our small angle X-ray scattering (SAXS) data of the SHU-complex. Taken together with our electrophoretic mobility shift assay (EMSA) results, a model is proposed for the SHU–protein complex coupled with DNA.Structured summary of protein interactions:PSY3 and CSM2 bind by X-ray crystallography (View interaction) PSY3, CSM2, Shu 1 and Shu 2 physically interact by x ray scattering (View interaction)     

Cell-cycle-dependent telomere elongation by telomerase in budding yeast

Telomeres are essential for the stability and complete replication of linear chromosomes. Telomere elongation by telomerase counteracts the telomere shortening due to the incomplete replication of chromosome ends by DNA polymerase. Telomere elongation is cell-cycle-regulated and coupled to DNA replication during S-phase. However, the molecular mechanisms that underlie such cell-cycle-dependent telomere elongation by telomerase remain largely unknown. Several aspects of telomere replication in budding yeast, including the modulation of telomere chromatin structure, telomere end processing, recruitment of telomere-binding proteins and telomerase complex to telomere as well as the coupling of DNA replication to telomere elongation during cell cycle progression will be discussed, and the potential roles of Cdk (cyclin-dependent kinase) in these processes will be illustrated.     

Sociobiology of the budding yeast

Social theory has provided a useful framework for research with microorganisms. Here I describe the advantages and possible risks of using a well-known model organism, the unicellular yeast Saccharomyces cerevisiae, for sociobiological research. I discuss the problems connected with clear classification of yeast behaviour based on the fitness-based Hamilton paradigm. Relevant traits include different types of communities, production of flocculins, invertase and toxins, and the presence of apoptosis.     

The centromere of budding yeast

Stable maintenance of genetic information during meiosis and mitosis is dependent on accurate chromosome transmission. The centromere is a key component of the segregational machinery that couples chromosomes with the spindle apparatus. Most of what is known about the structure and function of the centromeres has been derived from studies on yeast cells. In Saccharomyces cerevisiae, the centromere DNA requirements for mitotic centromere function have been defined and some of the proteins required for an active complex have been identified. Centromere DNA and the centromere proteins form a complex that has been studied extensively at the chromatin level. Finally, recent findings suggest that assembly and activation of the centromere are integrated in tethe cell cycle.     

Regulation of proliferation by the budding yeast Saccharomyces cerevisiae

Mutations in the budding yeast Saccharomyces cerevisiae define regulatory activities both for the mitotic cell cycle and for resumption of proliferation from the quiescent stationary-phase state. In each case, the regulation of proliferation occurs in the prereplicative interval that precedes the initiation of DNA replication. This regulation is particularly responsive to the nutrient environment and the biosynthetic capacity of the cell. Mutations in components of the cAMP-mediated effector pathway and in components of the biosynthetic machinery itself affect regulation of proliferation within the mitotic cell cycle. In the extreme case of nutrient starvation, cells cease proliferation and enter stationary phase. Mutations in newly defined genes prevent stationary-phase cells from reentering the mitotic cell cycle, but have no effect on proliferating cells. Thus stationary phase represents a unique developmental state, with requirements to resume proliferation that differ from those for the maintenance of proliferation in the mitotic cell cycle.